Bioactive Nitrogenous Secondary Metabolites from the Marine Sponge Genus Haliclona

Marine sponge genus Haliclona, one of the most prolific sources of natural products, contains over 600 species but only a small part of them had been classified and chemically investigated. On the basis of extensive literature search, this review firstly summarizes 112 nitrogenous secondary metabolites from classified and unclassified Haliclona sponges as well as from their symbiotic microorganisms. Most of these substances have only been found in Haliclona sponges, and display diverse bioactive properties with potential applications in new drug discovery.


Introduction
The marine environment is the largest treasure trove of creatures, including plants, animals and microorganisms. People never stop conducting chemical studies of marine organisms owing to their potential capability to produce bioactive secondary metabolites that are potential sources of leads for new drug development. So far over 34,000 articles about marine-derived natural products research have been published [1]. Marine sponges, which are immemorial organisms, are widely distributed around the world and comprise over ten thousand species, most of which them live in the sea, while only one percent are freshwater sponges [2]. These creatures are a great source of natural products with a broad spectrum of biological properties. Numerous sponge-derived chemicals, especial alkaloid compounds, display pharmacological effects, such as didemnin B, cytarabine, trabectedin, vidarabine, etc.
The marine sponge genus Haliclona belongs to the family Chalinidae, order Haplosclerida, class Demospongiae [3]. Its skeleton is made up of grids of single spines or a network of spongy fibrous branches without an epidermal skeleton [2]. The Haliclona genus consists of over 600 species distributed throughout the world [3]. However, only a few dozen specimens collected from the Pacific Ocean [4], north Indian Ocean [5], Atlantic Ocean [6], and Mediterranean Sea [7] have been chemically investigated. These studies suggest that Haliclona sponges are some of the most prolific producers of bioactive secondary metabolites. On the basis of a extensive literature search using SciFinder, this review firstly summarizes all nitrogenous substances from the marine sponge genus Haliclona and its symbiotic microbes.

Bioactive Alkaloids from Haliclona Genus
As many as 103 alkaloid secondary metabolites have been isolated and characterized from

Haliclona Densaspicula
Two novel alkaloids with six hexacyclic diamines, densanins A (13) and B (14) (Figure 3), were found in methanol extract of the sponge Haliclona densaspicula from Keomun Island (Korea) and their absolute chemical structures were determined by 1D and 2D NMR spectral analysis and Mosher reactions. Biological evaluation indicated that compounds 13 and 14 possess potent inhibitory effects on lipopolysaccharide-induced nitric oxide production in BV2 microglial cells with IC50 values of 1.05 and 2.14 μM, respectively [4,12].

Haliclona Baeri
There is only one report on a chemical study of Haliclona baeri collected from the coast of Jongbrii Province (Thailand) [8]. One new nitrogenated compound maleimide-5-oxime (1) along with one benzoic derivative and two tetillapyrones was separated from this sponge ( Figure 1). The follow-up bioassay tests suggested that compound 1 had weak cytotoxic activity against the human DAOY medulloblastoma cell line at 50 μg/mL [9].

Haliclona Densaspicula
Two novel alkaloids with six hexacyclic diamines, densanins A (13) and B (14) (Figure 3), were found in methanol extract of the sponge Haliclona densaspicula from Keomun Island (Korea) and their absolute chemical structures were determined by 1D and 2D NMR spectral analysis and Mosher reactions. Biological evaluation indicated that compounds 13 and 14 possess potent inhibitory effects on lipopolysaccharide-induced nitric oxide production in BV2 microglial cells with IC50 values of 1.05 and 2.14 μM, respectively [4,12].

Haliclona Densaspicula
Two novel alkaloids with six hexacyclic diamines, densanins A (13) and B (14) (Figure 3), were found in methanol extract of the sponge Haliclona densaspicula from Keomun Island (Korea) and their absolute chemical structures were determined by 1D and 2D NMR spectral analysis and Mosher reactions. Biological evaluation indicated that compounds 13 and 14 possess potent inhibitory effects on lipopolysaccharide-induced nitric oxide production in BV2 microglial cells with IC 50 values of 1.05 and 2.14 µM, respectively [4,12].

Haliclona Nigra
Fractionation of the aqueous extract of the marine sponge Haliclona nigra collected from northern coast of Papua New Guinea resulted in the discovery of two new hexapeptides, haligramides A (24) and B (25), together with waiakeamide (26) ( Figure 5) [21]. Their chemical structures and configurations were elucidated by extensive NMR analyses and oxidative reactions.

Other Haliclona Spps.
Until now, up to 71 nitrogenated compounds 33-103 have been discovered from unidentified marine sponge species of Haliclona. According to their chemical structures, these substances can be grouped into three classes, including 3-alkylpyridines, amides and depsipeptides, and miscellaneous alkaloids.

Other Haliclona Spps.
Until now, up to 71 nitrogenated compounds 33-103 have been discovered from unidentified marine sponge species of Haliclona. According to their chemical structures, these substances can be grouped into three classes, including 3-alkylpyridines, amides and depsipeptides, and miscellaneous alkaloids.

Bioactive Alkaloids from Haliclona-Derived Microbes
Marine sponges are important hosts for a large community of microorganisms, which are shown to be great producers of secondary metabolites [55]. However, only eight alkaloids 104-112 have been separated from Haliclona sponge-derived microbes until now ( Figure 10). Chemical analysis of the ethyl acetate extract of the strain Bacillus megaterium LC3CS2 symbiont of the sponge Haliclona oculata collected from Son Cha Peninsula (Vietnam) afforded three anti-microbial agents: 7,7-bis(3-indolyl)p-cresol (104), cyclo-(Pro-Leu) (105) and cyclo-(Pro-Val) (106) (Figure 10) [56]. These chemicals had antimicrobial activities against Vibrio vulnificus, V. parahaemolyticus and Trichophyton mentagrophytes with MIC values ranging from 0.05 to 5.0 µ g/mL. Compound 104, formerly obtained from a marine sponge Hyatell-derived microbe Vibrio sp. was shown to inhibit the growth of Bacillus cereus and Micrococcus luteus with MIC values of 0.5 and 0.005 µ g/mL, respectively [56,57]. Alantrypinone (107) along with lovastatin, methyl ester of lactone ring-opened monacolin K, terrein, territrems B and ergosterol was separated from a F62 fungal strain associated with the sponge Haliclona simulans collected from the South China Sea (Figure 10) [58]. Screening of symbiotic strains from the marine sponge Haliclona sp. collected from the sea shore of Tateyama city (Japan) led to the discovery of four new Streptomyces strains [59]. Later chemical investigation of strains GE-23 GE-26 and SC-24 afforded five new alkaloids JBIR-30, -34, -35, -39 and -40 (108-112) ( Figure 10). However, none of these compounds had potent cytotoxic effects on human cervical carcinoma HeLa cells and malignant pleural mesothelioma ACC-MESO-1 cells.

Conclusions
In summary, 112 nitrogenous secondary metabolites have been isolated and characterized from the marine sponge genus Haliclona and its derived microbes till now. Only five alkaloids (compounds 16, 22, 23, 91 and 104) were separated from other organisms. Therefore, this indicates that Haliclona sponges are some of the most prolific sources of exclusive bioactive alkaloids despite the fact only a handful of classified species had been chemically investigated. It is well-known that marine organisms have served as a primary source of bioactive natural products during the past several decades. Nowadays, however, a rapid decrease in the speed of discovery of new compounds from Nature strongly necessitates new research strategies and approaches. Microorganisms are ubiquitous in the ocean owing to their stronger adaptability. During long co-evolution with marine sponges, symbiotic microbes maybe play important physiological and ecological roles in promoting host growth and increasing the resistance to predators and omnivores by excreting toxic metabolites. Therefore, more efforts should be made to explore and identify unknown Haliclona sponges and their derived symbiotic microbes and to carry out chemical studies for the discovery of novel therapeutical agents.

Conflicts of Interest:
The authors declare no conflict of interest.